Haematological profile of malaria patients with G6PD and PKLR variants (erythrocytic enzymopathies): a cross-sectional study in Thailand

Background Glucose 6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PKLR) deficiencies are common causes of erythrocyte haemolysis in the presence of antimalarial drugs such as primaquine and tafenoquine. The present study aimed to elucidate such an association by thoroughly investigating the haematological indices in malaria patients with G6PD and PKLRR41Q variants. Methods Blood samples from 255 malaria patients from Thailand, Myanmar, Laos, and Cambodia were collected to determine haematological profile, G6PD enzyme activity and G6PD deficiency variants. The multivariate analysis was performed to investigate the association between anaemia and G6PD MahidolG487A, the most common mutation in this study. Results The prevalence of G6PD deficiency was 11.1% (27/244) in males and 9.1% (1/11) in female. The MAFs of the G6PD MahidolG487A and PKLRR41Q variants were 7.1% and 2.6%, respectively. Compared with patients with wildtype G6PD after controlling for haemoglobinopathies, G6PD-deficient patients with hemizygous and homozygous G6PD MahidolG487A exhibited anaemia with low levels of haemoglobin (11.16 ± 2.65 g/dl, p = 0.041). These patients also exhibited high levels of reticulocytes (3.60%). The median value of G6PD activity before treatment (Day 0) was significantly lower than that of after treatment (Day 28) (5.51 ± 2.54 U/g Hb vs. 6.68 ± 2.45 U/g Hb; p < 0.001). Reticulocyte levels on Day 28 were significantly increased compared to that of on Day 0 (2.14 ± 0.92% vs 1.57 ± 1.06%; p < 0.001). PKLRR41Q had no correlation with anaemia in malaria patients. The risk of anaemia inpatients with G6PD MahidolG487A was higher than wildtype patients (OR = 3.48, CI% 1.24–9.75, p = 0.018). Univariate and multivariate analyses confirmed that G6PD MahidolG487A independently associated with anaemia (< 11 g/dl) after adjusted by age, gender, Plasmodium species, parasite density, PKLRR41Q, and haemoglobinopathies (p < 0.001). Conclusions This study revealed that malaria patients with G6PD MahidolG487A, but not with PKLRR41Q, had anaemia during infection. As a compensatory response to haemolytic anaemia after malaria infection, these patients generated more reticulocytes. The findings emphasize the effect of host genetic background on haemolytic anaemia and the importance of screening patients for erythrocyte enzymopathies and related mutations prior to anti-malarial therapy. Supplementary Information The online version contains supplementary material available at 10.1186/s12936-022-04267-7.


Background
Glucose 6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and pyruvate kinase (PKLR; EC:2.7.1.40) deficiencies are the most common hereditary metabolic disorders affecting red blood cells [1,2]. G6PD deficiency triggers haemolytic anaemia in states of oxidative stress because deficient erythrocytes contain low levels of NADPH, which is required for maintaining cellular redox homeostasis through glutathione recycling [2]. Millions of people worldwide, mostly in Africa, the Mediterranean, the Middle East, and Asia, are affected by this condition. G6PD deficiency is caused by mutations in the G6PD gene on chromosome X. Genetically, males are either G6PD deficient or G6PD normal, while females can be homozygous with G6PD deficiency (mutations are present on both X chromosomes) or heterozygous (one X chromosome is affected) or G6PD normal. The frequency of G6PD status follows the Hardy Weinberg equilibrium. This makes heterozygous females are more common than hemizygous males and homozygous females are the least common [2].
Approximately 186 G6PD mutations, most of which are point mutations, have been documented [3]. Each mutation has different clinical phenotypes and distinctive geographical and ethnic distributions [4]. Recently, G6PD Mahidol G487A , a common Southeast Asian mutation, has been reported to reduce Plasmodium vivax density [5]. Additionally, a study in Afghanistan has demonstrates that G6PD deficiency protects against P. vivax clinical disease [6]. Even though G6PD deficiency provides clinical protection against Plasmodium spp., G6PD-deficient patients are susceptible to haemolytic anaemia when exposed to active and toxic metabolites of primaquine (PQ) and tafenoquine (TQ) [7][8][9]. PQ and TQ are anti-malarial drugs that reduce Plasmodium falciparum gametocytes for transmission and preventing the relapse of P. vivax and Plasmodium ovale malaria [8,9]. In 2013, Howes et al. published the spatial distribution of G6PD deficiency and its mutations in malaria-endemic areas around the globe to support the safe use of PQ and TQ [10]. The diagnosis of G6PD deficiency and molecular genotyping of G6PD in malaria patients prior to PQ and TQ administration are necessary to prevent adverse outcomes [8].
PK deficiency (PKD), the second most common enzyme deficiency, causes haemolytic anaemia worldwide with an estimated prevalence of 0.005% (1/20,000) in the Caucasian population. The prevalence of PKD in the European population was estimated to be less than 0.05% (5/10,000) [1], 3.4% in the Hong Kong population and 2.2% in Chinese infants [11,12]. The prevalence of PKD in Southeast Asian countries has yet to be determined. PKD is caused by loss-of-function mutations in PK predominantly expressed in the liver and red blood cells (PKLR). More than 150 mutations of PKLR have been reported [13]. Evidence in murine models has suggested that PKD confers a protective effect against malaria [14]. Recently, a novel point mutation (161A > G) resulting in an amino acid change at residue 41 from arginine (R), which is highly conserved in the PK family, to glutamine (Q) (R41Q) in the N-terminus of PK has been reported [15]. However, the haematological parameters in malaria patients with G6PD and PKLR R41Q mutations have not been thoroughly investigated. The main aim of the present study was to examine the haematological profiles in malaria patients with G6PD or PKLR mutations.

Study subjects and sample collection
The study protocol was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (Bangkok, Thailand) (COA No. 040/2013, IRB No. 459/55). All patients were screened by passive case detection (PCD) protocol and provided written informed consent prior to enrollment in this study. A total of 255 uncomplicated malaria patients who were admitted to the Hospital for Tropical Diseases in Thailand during 2011-2012 with blood slide positivity for Plasmodium spp. and had no history of anti-malarial drug treatment 2 weeks prior were recruited for this study. Blood samples were collected at the Hospital for Tropical Diseases in Bangkok, Thailand and transferred on ice to a research laboratory at the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand within an hour for immediate measurement of G6PD activity and haematological parameters. Complete blood count (CBC) was measured using an BC-6800 Auto Hematology Analyzer (Mindray Medical International, China).

Identify Plasmodium spp.
Giemsa staining of thick and thin blood smears prepared from finger pricks was examined every 12 h from initiation of treatment until they were negative. Blood smears were examined daily until patients were discharged. Plasmodium spp. were identified under Keywords: G6PD deficiency, Pyruvate kinase, Erythrocyte enzymopathy, G6PD Mahidol, Thailand, Southeast Asian, Plasmodium falciparum, Plasmodium vivax a microscope by an independent parasitologist at the Hospital for Tropical Diseases and further confirmed by polymerase chain reaction (PCR)-based analysis [16].

Measurement of G6PD activity
G6PD activity in the blood of malaria patients was measured in triplicate along with the normal and G6PD-deficient controls (G6888, G5888; Trinity Biotech, Ireland) using a quantitative assay kit for G6PD (Trinity Biotech, Ireland) prior to treatment and repeated weekly until patients were discharged. This assay measured NADPH production by G6PD in the blood of patients in parallel with positive and negative controls. Detection was carried out at a wavelength of 340 nm. The haemoglobin level was measured using Hb201 (HemoCue, Sweden) and used to calculate G6PD activity. Leftover blood samples were kept at − 20 °C for molecular typing.

Statistical analysis
All statistical analyses were performed using SPSS version 22 (IBM SPSS software, IL, USA). Data are expressed as percentages, median ± interquartile range (IQR), and mean ± standard deviation (SD). Following a computation approach reported previously, the adjusted male median (AMM) of G6PD activity was used to determine the cut-off values for G6PD deficiency [7].
The AMM values were defined as 100% activity of all male subjects after removing subjects with severe G6PD activity (≤ 10% of the overall median G6PD activity). The cut-off points for G6PD deficiency, G6PD intermediate (mild deficiency), and normal were median values less than 30%, 30 to 70%, and over 70% of the AMM, respectively. The G6PD activity of patients before (Day 0) and after treatment (Day 28) were compared using the Wilcoxon signed-rank test. The two-tailed Student's t-test was used to analyse the differences in quantitative variables. Haemoglobin less than 11 g/dl is considered anaemia [21]. The risk of anaemia in G6PD status and mutations was analysed by odd ratio (OR). In a study of the association between haematological parameters, G6PD deficiency/G6PD normal and G6PD Mahidol G487A / G6PD wildtype, a multiple linear regression was performed adjusted for age, gender, parasite species, parasite density, and haemoglobinopathies. A statistically significant difference was defined as two-sided with a p-value less than 0.05.  Table 1. These patients were from malaria-endemic areas, including the Thailand-Myanmar border (N = 25), Thailand-Cambodia border (N = 5) and several provinces of Thailand (N = 225), as described in a previous report [16] (Fig. 1). Two hundred forty-four patients (95.7%) were male, and eleven patients (4.3%) were female. The skewness of the gender ratio was influenced by male labor migration. The mean age of all patients was 27.94 ± 9.93 years (range 14-60 years). The numbers of patients infected with P. falciparum and P. vivax were 106 (41.6%) and 145 (56.9%), respectively. Three patients (1.2%) had coinfection of P. falciparum and P. vivax. One patient (0.4%) was infected with Plasmodium malariae.

Demographic data and prevalence of G6PD and PKLR R41Q mutations in malaria patients
The overall median value of G6PD activity in this cohort (n = 255) was 5.66 ± 2.55 U/g Hb (median ± IQR), ranging from 0.00 to 14.59 U/g Hb. The median values of G6PD activity in males (n = 244) and in females (n = 11) were 5.68 ± 2.49 and 4.80 ± 3.10 U/g Hb, ranging from 0.00 to 14.59 U/g Hb and 1.04 to 7.95 U/g Hb, respectively. The adjusted male median (AMM) G6PD activity in G6PD normal was 5.77 U/g Hb. The cut-off values for G6PD deficiency and G6PD intermediate were < 1.73 U/g Hb (< 30% of the AMM) and 4.04 U/g Hb (30-70% of the AMM), respectively (Fig. 2). G6PD activity exhibited bimodal distribution in males and normal distribution in females. The median values of G6PD activity in G6PD deficiency and G6PD intermediate were 0.49 ± 0.39 U/g Hb (range from 0.00 to 1.04 U/g Hb) and 3.59 ± 0.82 U/g Hb (range from 1.86 to 4.04 U/g Hb) (Fig. 2). According to these cut-off values, 27 male (11.1%; of a total of 244) and 1 female (9.1%; of a total of 11) patients were identified as G6PD deficient. The prevalence of G6PD intermediate in this study was 5.7% (14/ 244) in males and 27.3% (3/ 11) in females. Of 45 patients with G6PD deficiency and    to 0.66 U/g Hb), respectively. G6PD activity levels in 1 hemizygous male with G6PD Kaiping G1388A , 1 hemizygous male with G6PD Aures T143C , and 1 homozygous female with G6PD Mahidol G487A were 1.02 U/g Hb, 0.40 U/g Hb, and 1.04 U/g Hb, respectively. The PKLR R41Q mutation was detected in 12 patients (4.7%). These patients were from Thailand and Thailand borders (Fig. 1). The number of patients with PKLR R41Q who were infected with P. falciparum and P. vivax was equal. As shown in Table 1, PKLR R41Q was detected in malaria patients with an MAF of 2.6% in the study population (13 of 510), 2.6% in individuals from Myanmar (10 of 378), 2.9% in Thais (2 of 70) and 6.3% in Cambodians (1 of 16).

Haematological profiles of malaria patients with G6PD and PKLR mutations
At the first visit prior malaria treatment, malaria patients with G6PD deficiency (n = 28), compared to malaria patients with normal G6PD activity levels (n = 209), exhibited a significant decrease in the haemoglobin levels (11.03 ± 2.51 g/dl vs. 12.65 ± 1.97 g/ dl; p = 0.003). These patients also had a significant increase in the reticulocyte count (2.80 ± 2.05% vs. 1.49 ± 1.07%; p = 0.005). Malaria patients with G6PD Mahidol G487A mutation (n = 17) compared to wildtype patients without common Southeast Asian (SEA) mutations including the G6PD Mahidol G487A (n = 215) exhibited a significant decrease in haemoglobin levels (11.16 ± 2.65 g/dl vs. 12.66 ± 1.92 g/dl; p = 0.041). These patients also had an increase of reticulocyte levels (3.61 ± 2.44% vs. 1.47 ± 1.05%; p = 0.008) ( Table 2). There were not statistically differences in malaria patients with PKLR R41Q compared to those with wildtype. The coexistence of G6PD deficiency and thalassaemia/ haemoglobinopathies is very common in this region. One hundred twenty-nine malaria patients with thalassaemia and haemoglobinopathies were found in this study population. After excluding these patients, an association between G6PD deficiency and anaemia in malaria patients was found. The data were shown in Additional file 1: Table S1.

Discussion
Malaria infection causes haemolysis of infected erythrocytes. PQ and TQ, 8-aminoquinoline, are essential anti-malarial drugs commonly used for radical cure of P. vivax infections. It also reduces transmission of P. falciparum. However, PQ and TQ cause acute haemolytic complications in patients with G6PD deficiency. Although there have been many reports of G6PD deficiency status and its mutations in malaria patients living in the Southeast Asia, data on the haematological parameters of G6PD and other erythrocytic mutations including PK in malaria patients are limited. These data may be beneficial for the administration of anti-malarial treatment especially PQ and TQ prescription.
The overall frequency of patients with P. vivax infection was slightly higher than that of with P. falciparum infection, confirming that the frequencies of P. vivax and P. falciparum-infected cases are approximately equal, with high chances of coinfection in the international border between the territory of Myanmar and the western region of Thailand [22,23]. Overall, 28 (11.0%) participants were G6PD deficient, which was presented as  11.1% (27/244) of males and 9.1% (1/11) of females. This finding is consistent with previous studies reporting that G6PD deficiency was found in approximately 10.0-13.7% of the male population of these ethnic groups [24,25]. Although, the prevalence of G6PD deficiency in females (9.1%) in this population was higher than that reported previously (5.3%) [26], as a result of the small number of the female patients, the frequency of G6PD mutation in females follows Hardy-Weinberg equilibrium. Based on the population wide AMM, a total of 17 individuals (6.7%) exhibited intermediate G6PD activity, which was present in 5.7% (14/244) of males and 27.3% (3/11) of females. According to the results of this study and previous report, G6PD deficiency was more common in males than in females whereas intermediate was more common in females than in males [27]. These results showed that the G6PD Mahidol G487A mutation was more common among individuals from Myanmar, in Thai, and in Karen malaria patients, whereas G6PD Viangchan G871A mutation was more common among Thai and Cambodian malaria patients. In general, G6PD Viangchan G871A is more common in Thai people than G6PD Mahidol G487A [26]. Genetic admixture could explain the equal prevalence of G6PD Viangchan G871A and G6PD Mahidol G487A in this Thai population. The G6PD Mahidol G487A mutation in malaria patients had an MAF of 7.1% in the study population (18 of 255), 7.4% in individuals from Myanmar (14 of 189), 5.7% in Thais (2 of 35) and 12.5% in Karen (2 of 16). These findings agreed with the spatial distribution of G6PD deficient mutations in the Southeast Asia, where G6PD Mahidol G487A and G6PD Viangchan G871A mutations are commonly observed on the western and eastern Indochina Peninsula, respectively [5,17,26,28]. Although the G6PD genotype is a key factor of enzyme activity [29], some G6PD-deficient patients were unable to detect any mutations in G6PD coding regions. This could be explained by the methylation of CpG or CpNpG islands on G6PD promotor, resulting in gene silencing [30,31]. Another possibility is that the presence of mutation in the 5'untranslated region (UTR) that has been reported to reduce enzyme activity [32].
The frequency of PK in this Southeast Asian population was comparable to what was reported by van Bruggen et al., who found this mutation in 13 out of 340 healthy unrelated Southeast Asian subjects with an MAF of 3.2% in individuals from Myanmar, 1% in Thais, 1.5% in Cambodians, 1.8% in Laotians, and 2.9% in Mons [15]. Possible contributing factors for the discrepancy between these findings include the differences in population size, homogeneity within each ethnic group and the place of origin of each subject (malaria vs non-malaria endemic areas).
Correlations between altered G6PD activity due to mutations in malaria patients and haematological phenotypes prior to treatment with anti-malarial drugs have not been well studied. Based on the International Classification of Diseases, 11th Revision (ICD-11) considering classification of G6PD deficiency under haemolytic anaemias (code: 3A10.00) [33], the data demonstrated that malaria patients with G6PD deficiency prior to treatment, particularly the G6PD Mahidol G487A mutation, displayed signs of haemolytic anaemia, including low haemoglobin, RBC count, haematocrit, and high reticulocyte count. However, this study showed no signs of haemolytic anaemia in other G6PD and PKLR R41Q mutations. This is possibly due to the small number of patients enrolled, which limit the chance to detect haemolytic anaemia in malaria patients carrying G6PD Aures T143C , G6PD Viangchan G871A and G6PD Kaiping G1388A . According to the World Health Organization (WHO), G6PD variants are categorized based on the degree of enzyme deficiency and severity of haemolysis. G6PD Mahidol G487A and G6PD Aures T143C are in a class III mutation (moderately deficient) and G6PD Viangchan G871A and G6PD Kaiping G1388A are in a class II mutation (severely deficient) [3]. Increased G6PD activity levels after treatment in G6PD intermediate and normal groups was associated with reticulocytosis. The underlying mechanism for this phenomenon includes a post-treatment response of the bone marrow, which is suppressed during malaria infection [34][35][36][37][38][39].
G6PD Mahidol G487A was an independent risk factor for anaemia based on age, gender, parasite species, parasite density, PKLR R41Q , thalassaemia, and haemoglobinopathies. G6PD-deficient RBCs are exposed to oxidative stress caused by active neutrophilproduced ROS [40], leading to a decline in haemoglobin levels and generates reticulocytes [41]. According to the in-silico study by Bharti et al., G6PD enzymes with the Mahidol 487A mutation lose their crucial catalytic interaction with substrate [42]. In addition, Boonyuen et al. have reported that G6PD Mahidol G487A causes a local conformational change and affects backbone folding. This results in a reduction in thermostability in the absence or presence of NADP + and a reduction in K cat , thereby reducing catalytic efficiency [42,43]. The ability of erythrocytes to produce NADPH is diminished. NADPH is a reducing cofactor of glutathione reductase (GR), which reduces oxidized glutathione (GSSG) to reduced glutathione (GSH). GSH maintains the reduced state of the sulfhydryl group of haemoglobin and membrane proteins. In erythrocytes with the G6PD Mahidol 487A mutation, oxidation of membrane proteins causes the cells to rigid, nondeformable, and finally haemolysis. A recent report has indicated that patients